Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor γ (PPARγ) Function

Abstract

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1).A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor ␥ (PPAR␥) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cellbased transcription. Several additional amino acids in helix 4 of the PPAR␥LBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.Nuclear receptors are a large family of ligand-activated transcription factors that control important cell functions. Nuclear receptors contain a central DNA binding domain and a COOHterminal ligand binding domain (LBD) 1 with an activation function domain (AF2). Ligand-induced transactivation is mediated through interactions with members of a growing family of coactivator proteins, such as CBP (1-4), SRC-1 (3, 5-9), PGC-1(10), PBP (11, 12), TRAP220 (13), and ARA70 (14). A short sequence motif LXXLL which is shared by most coactivators has been shown to be required to mediate the ligandinduced receptor-coactivator interaction through AF2 (15)(16)(17). Mechanisms that confer the specificity for the nuclear receptorcoactivator interaction have become a central focus of investigation in this field. Different nuclear receptors have been found to preferentially interact with different coactivators and corepressors (18), suggesting that interactions besides LXXLL-AF2 binding per se play important roles. Darimont et al. (16) and McInerney et al. (17) have shown that the amino acids immediately adjacent the LXXLL motifs impart specific binding to selected nuclear receptors. Here, we identified the LBD residues of PPAR␥ that are required for both CBP and SRC-1 binding; in addition, we also identified several amino acids that differentially affect binding to CBP and SRC-1. These results suggest that certain residues within the LBD of nuclear receptors have an important role in mediating functional specificity by allowing for differential interactions with individual coactivators.

The RXRα C-terminus T462 is a NMR sensor for coactivator peptide binding

“…It is difficult to directly measure such a conformational exchange due to the loss of AF-2 resonances in the ternary complex. Similar effect on the AF-2 helix of a PPARγ LBD ternary complex has been reported [14], suggesting that AF-2 helix line broadening may be a general phenomenon of a NR LBD-ligand-coactivator peptide ternary complex in solution. It is important to determine whether this phenomenon can be extended to a RXR heterodimer.…”

Salt-inducible kinase 2 links transcriptional coactivator p300 phosphorylation to the prevention of ChREBP-dependent hepatic steatosis in mice

“…previously described to be important for the function of several transcription factors, such as PPARγ and FOXO1 (43,44). Thus, it is possible that phosphorylation of p300 at Ser89 by SIK2 affects its interaction with a select set of substrates.…”

A chromatin perspective of adipogenesis

“…81 When pRb is inactivated by phosphorylation upon induction of differentiation, this complex is dissociated and PPARg is then free to interact with HATs CBP/p300 and activate transcription. 81,93,94 Downregulation of CBP/p300 expression significantly reduces adipocyte differentiation. 93,95 Similarly, coexpression of HAT SRC1 with PPARg enhances the transcriptional activity of the factor and adipogenesis, whereas coexpression of corepressor NCoR suppresses it.…”

“…81,93,94 Downregulation of CBP/p300 expression significantly reduces adipocyte differentiation. 93,95 Similarly, coexpression of HAT SRC1 with PPARg enhances the transcriptional activity of the factor and adipogenesis, whereas coexpression of corepressor NCoR suppresses it. 96 In 3T3-L1 cells, addition of PPARg ligands breaks the PPARg/ NCoR complex and results in activation of PPARg transcriptional activity.…”